Identification of functional variants is part of the end-game of genetic analysis or perhaps the real starting point to understand roles of genes in behaviors. Public databases are populated with >12 million sequence variants, mostly single nucleotide polymorphisms. However, most rare and uncommon (<0.05) variants are unknown, and functionality of most is unknown. We discovered polymorphisms in >50 neurogenetic candidate genes. Several alter function of the encoded protein, or gene expression. A rare, gain-of-function serotonin transporter variant Ile425Val leads to severe pathology including Asperger's syndrome, treatment resistant OCD, and anorexia nervosa, in two families in which it is segregating. In the promoter region of this gene the HTTLPR polymorphism alters transcription. At HTTLPR we described a common, functional allele (LA->LG), enabling us to detect linkage of the gain-of-function LA allele to OCD in two populations (Hu et al). Discovery of the new functional allele (LG) enhanced linkage studies of HTTLPR to behavioral phenotypes and intermediate phenotypes, as described later. The findings include linkage to SSRI treatment response of depressed patients, via the mechanism of treatment tolerability (Hu et al, 2007). Common HTR2C Ser23Cys and HTR2A Asn452His alleles were detected, shown to be functional (Lappalainen et al, Ozaki et al, Okada et al) and linked by others to clozapine responsive of schizophrenics. In first-episode schizophrenics, we helped show that a functional DRD2 promoter polymorphism influences antipsychotic response (Lencz et al, 2006). We first detected the OPRM1 Asn40Asp missense variant (Bergen et al). It was shown by others to be functional and linked by others, and recently by us (Anton et al, 2008), to naltrexone treatment response in alcoholics. Recently we traced linkages of NPY (Zhu et al, 2008), GCH1 (Tegeder et al) and DISC1 (Hodgkinson et al) to behavior to functional haplotypes and alleles. [unreadable] The association we demonstrated between a low expression Neuropeptide Y (NPY) haplotype and increased anxiety and emotionality is illustrative of the effect of functional genetic variation on multiple levels of brain function and on complex behavior. Genetically determined reduction of expression of this anxiolytic neuropeptide predicted reduced trait anxiety and liability to anxiety disorders and increased brain responses to emotional stiuli as shown by fMRI and to pain/stress as shown by C11-carfentanil PET (Zhou et al, Nature, 2008). [unreadable] We created a 1536 SNP Addictions Array enabling haplotype-based and candidate locus coverage of 130 genes, including genes in the domains of alcohol metabolism, stress/anxiety, monoamine function, and signaling. The array includes 186 ancestry informative markers (AIMs) selected on the basis that they differed 0.7 and 10-fold in frequency between at least two continental populations and balanced for continental populations. The AIMs were genotyped in 52 CEPH reference populations, representing >1000 individuals, enabling us to derive ancestry factor scores for each individual in our datasets. The factor scores are used as covariates, ruling out or correcting for effects of ethnic stratification. Use of the array by Extramural investigators was facilitated by our performing genotyping such that 25,000 individuals were genotyped from multiple study samples including Yale, Emory University, the Rockefeller University, Columbia University, University of Colorado, UCSD, Medical College of Virginia, Washington University, and NIDCR (Hodgkinson et al, 2008). [unreadable] We detected loci influencing alcoholism by whole genome linkage analysis and followed up several. At the Chr 4 GABAA subunit cluster implicated in a Plains Indian linkage scan (Long et al) linkage disequilibrium to the GABAA alpha 2 gene was found by others. We replicated the alpha 2 LD finding and showed that the association to dependence was anxiety-mediated, or modulated (Enoch et al). Another GABAA gene cluster implicated in alcoholism, and alcohol response, is located on Chr 5. Within this cluster we reported linkage disequilibrium to alcoholism (Radel et al) and implicated the GABAA alpha 6 gene, where we discovered a missense variant associated with alcohol dependence and response to alcohol and diazepam (Iwata et al, Schuckit et al). We completed a genome scan in Plains Indians yielding genome-wide significant, or near-significant, linkage to an alcoholism-associated EEG trait, as described (AA000280-18).[unreadable] Intermediate phenotypes augment diagnosis by structured interview. Clinical subphenotyping enabled linkage of HTR1B to antisocial alcoholism (Lappalainen et al), serotonin transporte r (SLCA4) to anxiety (Mazzanti et al; Hariri et al), BDNF Val66Met to episodic memory (Egan et al), COMT Val158Met to anxiety (Enoch et al), executive cognition (Egan et al; Lipsky et al; Malhotra et al), and pain threshold (Zubieta et al; Diatchenko et al), and GTP cyclohydrolase to chronic pain and experimental pain response (Tegeder et al). In these studies brain imaging and cognitive measures play prominent roles. Frontal cognitive deficit is a risk factor in schizophrenia, alcoholism and other diseases. Dopamine generally enhances prefrontal cortical efficiency. Met158, a common COMT variant, leads to four-fold reduction in COMT activity. It is thus a candidate allele for cognitive function via effect on frontal dopamine. We found an allele-dosage relationship of Met158 to frontal cognitive function and diminished frontal cortical efficiency (Egan et al). The relationship to cognition is observed in populations differing in baseline cognitive function: schizophrenia, moderate-severe head injury (Lipsky et al), & controls (Malhotra et al). LNG proposed that Val158 has a counter-advantage: stress resiliency. In two populations Met158 predicted anxiety in women and decreased frontal EEG coherence (Enoch et al), and Met158 was associated with lower resiliency to pain/stress (Zubieta et al; Diatchenko et al). Met158 predicted inability to activate endomorphin release after pain/stress (Zubieta et al).Overall, effect sizes of genes in intermediate phenotypes is >3 whereas the modal effect size of 24 common alleles in seven complex diseases found by whole genome association was approximately 1.9 (Goldman and Ducci, 2007). [unreadable] Sampling framework and genetic structures are exploited to enhance detection of GxE effects, to achieve greater homogeneity of genetic background and exposures, and to enrich for exposures and outcomes. A Finnish dataset was ascertained from criminal alcoholic probands & thus enriched for Type II early onset alcoholism. SW Indian, Plains Indian, & Finnish datasets are derived from isolates, with psychiatrically interviewed controls available from source populations. An African American cocaine/opioid dependence dataset N=1000 was powerful for detecting GxE of childhood adversity and HTTLPR in adult suicidality (Roy et al, 2007) because of high rates of adversity and suicidality in substance dependence. An MAOA functional VNTR previously linked to dyscontrol via stress interaction was linked to outcomes of alcohol dependence and ASPD in American Indian women, of whom approximately half had been sexually abused as children (Ducci et al, 2008).[unreadable] The genome-wide integrative approach has been amplified by large scale genotyping enabling whole genome association, and next generation sequencing. We completed an Illumina 550k genome-wide scan for EEG variation associated with alcoholism and other psychiatric diseases and discovered seven independent genome-wide significant loci controlling variation in the EEG. Next-generation sequencing was used to analyze brain histone methylation and transcriptome changes resulting from early maternal deprivation in Rhesus macaques.